Este trabajo fue financiado por la Fundación Alemana para la Ciencia (DFG) y la Iniciativa de Excelencia de los gobiernos de Alemania federal y estatal.

1. Entrecruzamiento de material vegetal <ol><li> Cosecha de 1-2g de hojas de Arabidopsis (6 a 10 cm de diámetro roseta) en un tubo de plástico de 50 ml y completar hasta 40 ml con buffer de reticulación. </li><li> Asegúrese de que deja permanecer sumergidos, por ejemplo, el relleno a la derecha a medida filtro de esponja sobre la superficie de amortiguación. A continuación, coloque los tubos en un desecador. </li><li> Vacío infiltrarse durante 10 minutos. Añadir a cada tubo 2,5 ml de 2 M de glicina a dejar de reticulación, e invierta los tubos para garantizar la igualdad de la dispersión de la glicina. </li><li> Vacío para infiltrarse en otro 5 minutos y deseche el líquido, mientras que el corte de hojas en un tamiz. </li><li> Lavar las hojas dos veces con 1 litro de agua en un vaso de vidrio, y luego deja secar con toallas de papel. </li><li> Recoger las hojas en un tubo de plástico fresco y congelar en nitrógeno líquido. Tienda deja a -80 ° C hasta que el aislamiento de la cromatina. </li></ol> 2. Aislamiento de la cromatina <ol><li> Las hojas se molieron con un mortero y el pistilo que se mantuvieron en nitrógeno líquido hasta su uso. </li><li> Transferencia de polvo a un tubo de plástico de 50 ml y suspender en tampón de extracción de 30 ml # 1. </li><li> Incubar durante 15 min a 4 ° C en un agitador de arriba. </li><li> Filtrado suspensión a través de cuatro capas de Miracloth en un tubo de plástico fresco de 50 ml. </li><li> Giro de 20 minutos a 2.800 xg a 4 ° C. </li><li> Quitar el sobrenadante y suspender el precipitado con un pincel en tampón de extracción 1 ml # 2. Primero se debe agregar un pequeño volumen de tampón # 2, entonces suspender el precipitado con un pincel, y luego añadir el resto de buffer. </li><li> Transferencia de suspensión a un tubo de microcentrífuga 1,5 ml y girar durante 10 minutos a 12.000 xga 4 ° C. </li><li> Mientras tanto, prepare 2 ml-microcentrífuga tubos que contienen 1,5 ml de tampón de extracción # 3. </li><li> Aspirar el sobrenadante del tubo de hilar (paso 2.7) y suspender el precipitado en 300μL de tampón de extracción # 3. Primero se debe agregar un pequeño volumen de buffer # 3, suspender el precipitado con un pincel, y luego agregar el resto del búfer. </li><li> Cuidadosamente con pipeta el precipitado en suspensión (paso 2.9) en la parte superior del búfer de 1,5 ml de extracción # 3 preparada en la etapa 2.8. Asegúrese de que las dos fases no se mezclan. Giro de 1 hora a 16.000 xg a 4 ° C. </li><li> Aspirar el sobrenadante y suspender la cromatina pastilla con un pincel en 300μL de tampón de sonicación. </li><li> Sonicar cromatina con un sonotrodo, o en un baño de ultrasonidos, con un tamaño de ADN de aproximadamente 400bp. Cuando ultrasonidos, que la cromatina que no se calienta por encima de aproximadamente 30 ° C para proteger sensibles al calor enlaces cruzados. La duración del tratamiento con ultrasonidos debe determinarse experimentalmente, ya que varía considerablemente entre los dispositivos de ultrasonidos diferentes. Para la dirección, la configuración de dos dispositivos diferentes están disponibles: </li></ol> De ultrasonidos con una marca Bandelin sonotrodo exponer la cromatina en 0,6 ml tubo de microcentrífuga cinco veces a 20 explosiones con la configuración de un 25% de energía, ciclo = 50. Al hacerlo, muestra fría después de cada 20 explosiones º en un baño de hielo. De ultrasonidos con un baño de ultrasonidos Diagenode Bioruptor, llenar baño con agua y hielo y ultrasonidos diez veces durante 30 segundos en los tubos de 1,5 ml de microfuga con el ajuste &quot;alto&quot;. Después cada 30 segundos, las muestras frescas durante otros 30 segundos. <ol start="13"><li> Giro sonicado muestra durante 5 minutos a 16.000 ga 4 ° C para precipitar el material no disuelto. El sobrenadante se puede utilizar directamente para la inmunoprecipitación. Por otra parte, las muestras pueden ser de choque congelado en nitrógeno líquido y almacenadas a -80 ° C hasta su uso posterior. </li></ol> 3. Inmunoprecipitación <ol><li> Prepare de 2 ml que contienen tubos de microcentrífuga 40μL de proteína-A. Agarosa y 1,8 ml de anticuerpo de unión de amortiguación Añadir 200μL de la preparación de la cromatina (paso 2,13). </li><li> Incubar los tubos durante 1 hora en un agitador de arriba. </li><li> Deseche la proteína-A perlas de agarosa y el uso de sobrenadante de la inmunoprecipitación. </li><li> Eliminar una parte alícuota de 40μL-para determinar la concentración de la cromatina (&#39;input&#39;). </li><li> Añadir 30μL de proteína-A. Agarosa y la cantidad de modificación de anticuerpos específicos que se indica en la siguiente tabla de reactivos y equipos específicos para la suspensión 400μL cromatina sonica </li><li> Incubar durante 2,5 h o durante la noche en un agitador de arriba a 4 ° C. </li><li> Centrifugar durante 2 minutos en cuentas x 440 g. </li><li> Lávese las bolas pellet con 900μL de cada solución lavadora (véase la lista más abajo). Para cada buffer, cuentas de incubar en la memoria durante 10 minutos en un agitador de arriba a 4 ° C, y luego girar durante 2 minutos a 440 xg, desechar el sobrenadante y añadir el siguiente búfer. <ol><li> Baja en sal tampón de lavado </li><li> Elevado de sal tampón de lavado </li><li> LiCl tampón de lavado </li><li> TE tampón de lavado </li></ol></li><li> Después del último lavado, eliminar por completo cualquier resto del búfer. </li></ol> 4. De reticulación de las histonas y el ADN, y la purificación de ADN se precipitó <ol><li> Añadir 100μL de tampón de reticulación de la pastilla de agarosa y se muestra &quot;entrada&quot; de la 40μl de paso 3.4, la mezcla durante 5 minutos en un vórtex, las muestras de giro, y la INCUBate a 65 ° C durante la noche. </li><li> Purificar el ADN con el ADN comercial o kit de purificación de PCR. </li><li> Por lo general 5μl de una dilución 1:05 de la eluido de la columna final son suficientes para realizar convencional específica de locus cuantitativa en tiempo real RT-PCR (RT-qPCR). </li></ol> 5. Tabla de buffers <table border="1"><tbody><tr><td colspan="2"> Entrecruzamiento de búfer </td></tr><tr><td> Butirato sódico </td><td> 10 mM </td></tr><tr><td> Sacarosa </td><td> 400 mM </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10 mM </td></tr><tr><td> β-mercaptoetanol </td><td> 5mM </td></tr><tr><td> Formaldehído </td><td> 3% (v / v) </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Tampón de extracción # 1 </td></tr><tr><td> Butirato sódico </td><td> 10 mM </td></tr><tr><td> Sacarosa </td><td> 400 mM </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10 mM </td></tr><tr><td> β-mercaptoetanol </td><td> 5mM </td></tr><tr><td> Inhibidor de la proteasa completa </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Tampón de extracción # 2 </td></tr><tr><td> Butirato sódico </td><td> 10 mM </td></tr><tr><td> Sacarosa </td><td> 250 mm </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10 mM </td></tr><tr><td> β-mercaptoetanol </td><td> 5mM </td></tr><tr><td> MgCl2 </td><td> 10 mM </td></tr><tr><td> Triton X-100 </td><td> 1% (w / v) </td></tr><tr><td> Inhibidor de la proteasa completa </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Tampón de extracción # 3 </td></tr><tr><td> Butirato sódico </td><td> 10 mM </td></tr><tr><td> Sacarosa </td><td> 1,7 millones </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10 mM </td></tr><tr><td> β-mercaptoetanol </td><td> 5mM </td></tr><tr><td> MgCl2 </td><td> 2 mM </td></tr><tr><td> Triton X-100 </td><td> 0,15% (w / v) </td></tr><tr><td> Inhibidor de la proteasa completa </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Sonicación buffer </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 25 mM </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 5mM </td></tr><tr><td> SDS </td><td> 0,5% (w / v) </td></tr><tr><td> Inhibidor de la proteasa completa </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Unión de los anticuerpos de búfer </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 50 mM </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 1 mM </td></tr><tr><td> NaCl </td><td> 150 mM </td></tr><tr><td> Triton X-100 </td><td> 0,1% (w / v) </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Baja en sal tampón de lavado </td></tr><tr><td> NaCl </td><td> 150 mM </td></tr><tr><td> SDS </td><td> 0,1% (w / v) </td></tr><tr><td> Triton X-100 </td><td> 1% (w / v) </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 2 mM </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 20 mM </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Elevado de sal tampón de lavado </td></tr><tr><td> NaCl </td><td> 500 mm </td></tr><tr><td> SDS </td><td> 0,1% (w / v) </td></tr><tr><td> Triton X-100 </td><td> 1% (w / v) </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 2 mM </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 20 mM </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> LiCl tampón de lavado </td></tr><tr><td> Cloruro de litio </td><td> 250 mm </td></tr><tr><td> Nonidet-P40 </td><td> 1% (v / v) </td></tr><tr><td> Desoxicolato de sodio </td><td> 1% (w / v) </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 1 mM </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 20 mM </td></tr></tbody></table><table border="1"><tbody><tr><td> TE tampón de lavado </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 10 mM </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 1 mM </td></tr></tbody></table><table border="1"><tbody><tr><td> Decrosslinking buffer </td></tr><tr><td> Tris-HCl, pH 6,8 </td><td> 62,5 mm </td></tr><tr><td> NaCl </td><td> 200 mM </td></tr><tr><td> SDS </td><td> 2% (w / v) </td></tr><tr><td> TDT </td><td> 10 mM </td></tr></tbody></table> 6. Los resultados representativos: La expresión del gen de defensa de Arabidopsis thaliana que codifica una β PR2-1,3-glucanasa con actividad antimicrobiana 12, fue inducida por benzothiadiazole (BTH), un análogo sintético de la hormona vegetal ácido salicílico 3. La figura 1 muestra que la activación de PR2 expresión se asocia con un aumento en la acetilación de residuos de lisina de las histonas H3 y H4 en el promotor de PR2. Dado que la densidad disminuye nucleosoma durante la activación del gen 13, los valores de acetilación de histonas se normalizaron por la señal obtenida con un anticuerpo contra una región invariable de la histona 3. <img src="/files/ftp_upload/3096/3096fig1.jpg" alt="Figura 1"/> Figura 1 BTH induce la acetilación de histonas en el promotor de PR2 en Arabidopsis. Las plantas fueron tratadas con 100μM BTH o un control de polvo humectable. Después de 72 horas, se recogieron las hojas y la cromatina se aisló. La señal obtenida después de la precipitación con la acetilación de anticuerpos específicos (como se indica en la figura) se normalizó a la señal obtenida por precipitación con un anticuerpo a un dominio de los idiomas de la histona H3. Cromatina precipitado se cuantificó por RT-PCR cuantitativa. Los datos están estandarizados para los niveles de modificación de las histonas en la ausencia de tratamiento BTH.

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ordered+histone+modifications+are+associated+with+transcriptional+poising+and+activation+of+the+phaseolin+promoter.">Ordered histone modifications are associated with transcriptional poising and activation of the phaseolin promoter.</a> Plant Cell. 18, 119-132 (2006).</li><li>Offermann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illumination+is+necessary+and+sufficient+to+induce+histone+acetylation+independent+of+transcriptional+activity+at+the+C4-specific+phosphoenolpyruvate+carboxylase+promoter+in+maize.">Illumination is necessary and sufficient to induce histone acetylation independent of transcriptional activity at the C4-specific phosphoenolpyruvate carboxylase promoter in maize.</a> Plant Physiol. 141, 1078-1088 (2006).</li><li>Deng, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+histone+acetyltransferase+AtHAC1+in+the+regulation+of+flowering+time+via+repression+of+FLOWERING+LOCUS+C+in+Arabidopsis.">Involvement of the histone acetyltransferase AtHAC1 in the regulation of flowering time via repression of FLOWERING LOCUS C in Arabidopsis.</a> Plant Physiol. 143, 1660-1668 (2007).</li><li>Benhamed, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+GCN5,+HD1,+and+TAF1/HAF2+interact+to+regulate+histone+acetylation+required+for+light-responsive+gene+expression.">Arabidopsis GCN5, HD1, and TAF1/HAF2 interact to regulate histone acetylation required for light-responsive gene expression.</a> Plant Cell. 18, 2893-2903 (2006).</li><li>Bowler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+techniques+for+plant+cells.">Chromatin techniques for plant cells.</a> Plant Journal. 39, 776-789 (2004).</li><li>Orlando, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+chromosomal+proteins+in+vivo+by+formaldehyde+decrosslinked-chromatin+immunoprecipitation.">Mapping chromosomal proteins in vivo by formaldehyde decrosslinked-chromatin immunoprecipitation.</a> Trends Biochem. 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No hay conflictos de interés declarado.

Pasos más importantes en el protocolo son el entrecruzamiento de ADN y las histonas, el tipo y cantidad de material de la hoja, la molienda de la materia, y la sonicación de la cromatina. Para una discusión más detallada de estos temas, ver refs. 9 y 10. Sonicación y el entrecruzamiento: Es importante para interrumpir la cromatina durante la sonicación a los fragmentos de alrededor de 400bp. Sonicación exhaustiva va a destruir la cromatina mediante la interrupción de ADN y las histonas, mientras que la sonicación insuficiente dejará largos fragmentos de la cromatina. Estos afectan a la especificidad del método, porque las modificaciones de las histonas distal del locus pruebas no pueden ser discriminados de modificaciones locales. La eficiencia es la mejor prueba de sonicación mediante la recopilación de la cromatina 20μL de las muestras antes y después del tratamiento con ultrasonidos, de reticulación del ADN y las histonas, aislando el ADN y la determinación de la longitud del ADN cortado por electroforesis en gel de agarosa. RNAsa debe ser añadido a las muestras antes de la electroforesis, ya que la preparación de la cromatina todavía contiene grandes cantidades de ARN en esta etapa de purificación que pueda interferir con la visualización de ADN fragmentado. Las muestras son útiles para la inmunoprecipitación de cromatina si el ADN de la cromatina no esquilada es más largo que 10kbp, mientras que el ADN de la cromatina cortado formas una prueba con una intensidad máxima de la señal alrededor de 400bp de ADN. Nosotros usamos el 3% (v / v) de formaldehído para la reticulación de la cromatina en hojas intactas, pero el 1% (v / v) de formaldehído puede ser suficiente en la mayoría de los casos. En nuestras manos, las bajas concentraciones de formaldehído permitir tiempos de sonicación más cortos y las señales de fondo en la consiguiente reacción de PCR. Sin embargo, una cantidad insuficiente de formaldehído a menudo resultan en una baja reproducibilidad de la señal de PCR entre experimentos independientes. Esto puede ser debido a la penetración insuficiente de las hojas con formaldehído en concentraciones bajas. Asegúrese de que el intercambio de sus acciones de formaldehído con frecuencia como el compuesto tiende a polimerizar con el tiempo. Las soluciones de formaldehído son estables sólo durante aproximadamente un mes, y esta época no es prolongada por la estabilización de metanol. Se recomienda probar diferentes concentraciones de formaldehído en la creación de condiciones experimentales. Cantidad de material vegetal y de molienda: Es importante para infiltrar pequeñas cantidades de material de la hoja en un gran volumen de tampón para evitar que las hojas se peguen entre sí. Hoja se pegue a menudo resulta en la infiltración insuficiente de formaldehído. Además, el material vegetal demasiada bloquear los poros del tejido Miracloth. La eficiencia de la molienda depende considerablemente en el uso de un mortero con el ajuste perfecto. En forma rutinaria molienda con un nitrógeno líquido refrigerado mortero en la ausencia de nitrógeno líquido adicional de tres veces durante 50 segundos hasta que el material se muele hasta obtener un polvo fino.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> Proteína A-agarosa </td><td> Roche </td><td> 11 134 515 001 </td><td> depende de la clase de anticuerpos </td></tr><tr><td> Proteína-G-agarosa </td><td> Roche </td><td> 11 243 233 001 </td><td> depende de la clase de anticuerpos </td></tr><tr><td> Anti-hyperacetyl-H4 </td><td> Millipore </td><td> 06-946 </td><td> hemos utilizado 5μL </td></tr><tr><td> Anti-acetil-H4K5 </td><td> Millipore </td><td> 07-327 </td><td> hemos utilizado 5μL </td></tr><tr><td> Anti-acetil-H4K8 </td><td> Millipore </td><td> 07-328 </td><td> hemos utilizado 5μL </td></tr><tr><td> Anti-acetil-H4K12 </td><td> Millipore </td><td> 07-595 </td><td> hemos utilizado 5μL </td></tr><tr><td> Anti-acetil-H4K16 </td><td> Millipore </td><td> 07-329 </td><td> hemos utilizado 5μL </td></tr><tr><td> Anti-acetil-H4K18 </td><td> Millipore </td><td> 07-354 </td><td> hemos utilizado 1μL </td></tr><tr><td> Anti-acetil-H3K9 </td><td> Millipore </td><td> 07-352 </td><td> hemos utilizado 5μL </td></tr><tr><td> Anti-acetil-H3K14 </td><td> Millipore </td><td> 07-353 </td><td> hemos utilizado 1μL </td></tr><tr><td> Anti-trimetil H3K4 </td><td> Diagenode </td><td> PAB-003-50 </td><td> hemos utilizado 5μL </td></tr><tr><td> Anti-dimetil-H3K4 </td><td> Millipore </td><td> 07-030 </td><td> hemos utilizado 5μL </td></tr><tr><td> Anti-monometil-H3K4 </td><td> Abcam </td><td> ab8895 </td><td> 2,5-10μL </td></tr><tr><td> Anti-H3 </td><td> Abcam </td><td> ab1791 </td><td> hemos utilizado para comprobar la densidad de 1μL histona </td></tr><tr><td> Bioruptor </td><td> Diagenode </td><td> UCD-200 A </td><td></td></tr><tr><td> MS72 sonotrodo </td><td> Bandelin </td><td></td><td></td></tr><tr><td> Miracloth </td><td> Calbiochem </td><td> 475855 </td><td></td></tr><tr><td> Inhibidor de la proteasa completa </td><td> Roche </td><td> 11836145001 </td><td></td></tr></tbody></table>

detection of histone modifications in plant leaves

Estructura de la cromatina es importante para la regulación de la expresión génica en las células eucariotas. En este proceso, las modificaciones de remodelación de la cromatina, la metilación del ADN, y covalentes en las colas amino-terminal de las histonas H3 y H4 juegan un papel esencial 1-2. Modificaciones de histonas H3 y H4 incluyen la metilación de la lisina y la arginina, la acetilación de la lisina, y la fosforilación de 1-2 residuos de serina. Estas modificaciones se relacionan tanto con la activación de genes, la represión, o en un estado preparado de gen que admite la activación más rápida y robusta de expresión después de la percepción de las señales apropiadas (microbio patrones moleculares asociados, la luz, hormonas, etc) 3-7. A continuación, presentamos un método para la detección fiable y sensible de determinadas modificaciones de la cromatina en los genes de plantas seleccionadas. La técnica se basa en el entrecruzamiento de (modificar) las histonas y el ADN con formaldehído 8,9, extracción y tratamiento con ultrasonidos de la cromatina, cromatina immunoprecipitation (CHIP) con la modificación de los anticuerpos específicos de 9,10, de reticulación de la histona-ADN complejos, y de genes específicos en tiempo real PCR cuantitativa. El enfoque ha demostrado ser útil para la detección de modificaciones de las histonas específicas asociadas a la fotosíntesis C 4 en el maíz 5,11 y la inmunidad sistémica en Arabidopsis 3.

Watch this Scientific Journal Video about Detection of Histone Modifications in Plant Leaves at JoVE.com

Detection of Histone Modifications in Plant Leaves

Un enfoque confiable y útil para detectar modificaciones de las histonas en los genes específicos de la planta se describe. El enfoque combina la cromatina immunoprecipitation (CHIP) y en tiempo real PCR cuantitativa. Que permite la detección de modificaciones de las histonas en los genes específicos con un papel en diversos procesos fisiológicos.

La detección de modificaciones de las histonas en hojas de las plantas

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

detection-of-histone-modifications-in-plant-leaves

Research

JoVE Journal

Biology

24.7K vistas.  RWTH Aachen University. Un enfoque confiable y útil para detectar modificaciones de las histonas en los genes específicos de la planta se describe. El enfoque combina la cromatina immunoprecipitation (CHIP) y en tiempo real PCR cuantitativa. Que permite la detección de modificaciones de las histonas en los genes específicos con un papel en diversos procesos fisiológicos.

Video: Detection of Histone Modifications in Plant Leaves

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

La detección de modificaciones post-traduccionales de los nativos intactos nucleosomas por ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Investigación

Biología

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

Detección de interacciones de proteínas en la planta mediante un Gateway Compatible Complementación fluorescencia bimoleculares (BiFC) del sistema

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Perfiles de Polímeros Glycan la pared celular vegetal utilizando microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Identificación de modificaciones post-traduccionales de la proteína vegetal Complejos

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

La técnica de infiltración-centrifugación para extracción de apoplástico fluido procedente de hojas de la planta Uso<em&gt; Phaseolus vulgaris</em&gt; Como ejemplo

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Alta resolución Cuantificación de celulosa cristalina acumulación en<em&gt; Arabidopsis</em&gt; Las raíces para monitorear la célula específica de tejido de pared Modificaciones

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

De alto rendimiento CRISPR vector de la construcción y caracterización de las modificaciones del ADN mediante la generación de raíces peludas tomate

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Flujo de trabajo completo para el análisis de las histonas modificaciones post-traduccionales El uso de espectrometría de masas de abajo hacia arriba: A partir de la histona Extracción de Análisis de Datos

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Identificación de nuevos reguladores de la transpiración de plantas mediante el cribado térmico de imágenes a gran escala en Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown.

This protocol outlines how a three-dimensional cell culture system can be used to model, treat, and analyze chromatin modifications in a near-physiological state.

Cuantificación a nivel global de modificaciones post-traduccionales de histonas en un modelo de cultivo celular 3D de tejido hepático

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid ...